The present invention relates to screening assays for the identification of inhibitors of the biological activity of macrophage migration inhibitory factor (MIF). In addition, the present invention relates to the organic molecules, proteins and peptides identified by the screening assay. In particular, the invention relates to the uses of such identified compounds in pharmacological compositions and methods for the treatment of various conditions involving cytokine-mediated toxicity, which include, but are not limited to shock, inflammation, graft-versus-host disease, and/or autoimmune diseases.
Infection by a variety of microorganisms including not only bacteria but also viruses, fungi, and parasites can induce septic shock, a multifaceted pathological condition characterized most prominently by deleterious hemodynamic changes and coagulopathy leading to multiple organ failure and often to death. In fact, the shock syndrome is more properly associated with the host""s response to invasion rather than just infection, as cancer and trauma, for instance, can also serve as initiators. In the case of infection by gram-negative bacteria, one of the best studied examples, it is believed that the appearance of bacterial endotoxins such as lipopolysaccharide (LPS) in the host bloodstream leads to the endogenous production of a variety of host factors that directly and indirectly mediate the toxicity of LPS, which itself is relatively innocuous for most cells. These host-derived mediators include many now well-recognized inflammatory cytokines and classical endocrine hormones in addition to a number of other endogenous factors such as leukotrienes and platelet activating factor. It is generally acknowledged, however, that the full cast of participants and each of their interrelated roles in the host response remains incompletely appreciated.
In general, those mediators that appear earlier in an invaded host are thought to trigger the release of later appearing factors. Also, many endogenous mediators not only exert direct effector functions at their target tissues, but also prime local and remote tissues for subsequent responses to other mediators. This interacting network of host factors has been termed the xe2x80x9ccytokine cascade.xe2x80x9d This term is meant to indicate the rapid extension and amplification of the host response in such a way that only one or a few initiating stimuli trigger the eventual release and participation of scores of endogenous mediators. Although a number of features of the host response are thought to assist in fighting off invasion, an overly robust or poorly modulated host response can rapidly accelerate to produce such profound alterations in host homeostasis at the cellular, tissue, and systemic levels that death may ensue within hours.
2.1. The Pituitary as a Source of Protective Cytokines
Recent studies suggest that the pituitary gland may produce factors that inhibit the production of endotoxin-induced cytokines and other mediators of the host response, such as TNFxcex1 and IL-1, and thus may serve as a source for potentially protective factors that may be used to treat shock and/or other inflammatory responses. (Suzuki et al., 1986, Am. J. Physiol. 250: E470-E474; Sternberg et al., 1989, Proc. Natl. Acad. Sci. USA 86: 2374-2378; Zuckerman et al., 1989, Eur. J. Immunol. 19: 301-305; Edwards III et al., 1991, Endocrinol. 128: 989-996; Edwards III et al., 1991b, Proc. Natl. Acad. Sci. USA 88: 2274-2277, Silverstein et al., 1991, J. Exp. Med. 173:357-365). In these studies, hypophysectomized mice (i.e., animals that have had their pituitary glands surgically removed) exhibited a markedly increased sensitivity to LPS injection relative to sham-operated control mice. In fact, the LPS LD100 for control mice was approximately 1-2 logs higher than that determined for the hypophysectomized mice, suggesting that the pituitary gland produces one or more factors that may act to increase the host animal""s ability to resist endotoxin challenge. Some of these studies implicate the involvement of ACTH and adrenocorticosteroids (e.g., Edwards III et al., 1991a and 1991b, supra); however, other data suggest the existence of other protective factors derived from the pituitary.
Very recently, murine macrophage migration inhibitory factor (MIF) was identified as an LPS-induced pituitary protein (Bernhagen et al., 1993, J. Cell. Biochem. Supplement 17B, Abstract E306). While it was hypothesized that MIF was one of the pituitary protective factors capable of counteracting the adverse effects of cytokines in endotoxemias, it was found that MIF actually exacerbates endotoxin-induced shock, and that inhibition of MIF activity can be used to treat otherwise lethal effects of cytokine-mediated activity (see Application Ser. No. 08/243,342, which is incorporated by reference herein in its entirety). As explained in the subsection below, MIF has been re-defined recently to be an anterior pituitary hormone, a macrophage cytokine, and a critical component of the host response to septic shock (Bernhagen et al., 1993, Nature 365:756-759; Calandra et al., 1994, J. Exp. Med. 179:1895-1902; Calandra et al., 1995, Nature 377:68-71). MIF exists preformed in various cell types and in the last few years has been fortuitously identified in several other organ systems and physiological contexts.
2.2. MIP: Macrophage Migration Inhibitory Factor
Although MIF was first described over 30 years ago as a T cell product that inhibits the random migration of guinea pig macrophages in an in vitro assay (George and Vaughan, 1962, Proc. Soc. Exp. Biol. Med. 111:514-521; Bloom and Bennett, 1966, Science 158: 80-82; David, 1966, Proc. Natl. Acad. Sci. USA 65: 72-77), the precise role of MIF in either local or systemic inflammatory responses had remained largely undefined. MIF has been reported to be associated with delayed-type hypersensitivity reactions (Bloom and Bennett, 1966, supra; David, 1966, supra), to be produced by lectin-activated T-cells (Weiser et al., 1981, J. Immunol. 126: 1958-1962), and to enhance macrophage adherence, phagocytosis and tumoricidal activity (Nathan et al., 1973, J. Exp. Med. 137: 275-288; Nathan et al., 1971, J. Exp. Med. 133: 1356-1376; Churchill et al., 1975, J. Immunol. 115: 781-785). Unfortunately, many of these studies used mixed culture supernatants that were shown later to contain other cytokines such as IFN-xcex3 and IL-4 that also have migration inhibitory activity (McInnes and Rennick, 1988, J. Exp. Med. 167: 598-611; Thurman et al., 1985, J. Immunol. 134: 305-309).
Recombinant human MIF was originally cloned from human T cells (Weiser et al., 1989, Proc. Natl. Acad. Sci. USA 86: 7522-7526), and although the biological activity profile of MIF is incompletely known and has been debated, MIF has been shown to activate blood-derived macrophages to kill intracellular parasites and tumor cells in vitro, to stimulate IL-1xcex2 and TNFxcex1 expression, and to induce nitric oxide synthesis (reviewed in Bernhagen et al., 1994, Biochemistry 33:14144-14155).
MIF has been described recently to be an anterior pituitary hormone and to be released from immune cells stimulated by low concentrations of glucocorticoids. Once secreted MIF acts to control, or counter-regulate, the immunosuppressive effects of glucocorticoids on inflammatory cytokine production and acts to modulate the potent anti-inflammatory properties of glucocorticoids which are necessarily produced as part of the host response to infection and tissue invasion (Calandra et al., 1995, Nature 377:68-71). MIF is a critical component of the immune system and acts to counter the immunosuppressive effect of glucocorticoids to regulate inflammation and immunity.
Despite the interest in MIF activity, the lack of any pharmacological inhibitors of MIF has continued to hinder the investigation of the precise biological role of this molecule. For example, in contrast to its expected function as a protective hypothalamus factor, MIF proved to exacerbate lethality in several models of septic shock, indicating that a robust MIF response within the cytokine cascade may be undesired in a number of clinical settings. Given the critical role that MIF plays in cytokine-mediated toxicity in the host immune response, it would be of interest to use inhibitors of MIF to treat various conditions involving cytokine-mediated toxicity, including shock, inflammation, graft versus host disease, and/or immune diseases. However, other than antibodies directed against the MIF protein, there are no known exogenous inhibitors of MIF immunologic activity. Furthermore, the lack of defined cellular and biochemical activities of MIF has made it very difficult for researchers to develop an assay to specifically measure and quantitate MIF immunologic activity and identify MIF inhibitors. The lack of an in vitro assay to identify MIF inhibitors has hampered the investigation of the precise biological profile of this molecule in the immune response and the development of a treatment for disorders related to cytokine-mediated toxicity.
The present invention relates to a rapid, quantitative and specific assay for screening test compounds, such as drugs, ligands (natural or synthetic), proteins, peptides and small organic molecules for their ability to inhibit the biological activity of macrophage migration inhibitory factor (MIF).
The present invention also relates to the drugs, ligands, proteins, peptides and small organic molecules identified by the screening assay of the present invention as capable of inhibiting biological activities of MIF.
In particular, the invention relates to methods of using such identified compounds in pharmaceutical compositions for the treatment of various conditions involving cytokine-mediated toxicity, which include, but are not limited to shock, inflammation, graft-versus-host disease, and/or autoimmune diseases.
The invention is based, in part, on the surprising discovery and demonstration that MIF catalyzes a tautomerization reaction, and that assays for inhibitors of this tautomerase activity can be used to identify compounds that inhibit other biological activities of MIF. The enzymatic activity of MIF was uncovered during an investigation of the biochemical pathways of melanogenesis. The late stages of melanin biosynthesis involve the enzymatic conversion of L-2-carboxy-2,3-dihydroindole-5,6-quinone (L-dopachrome) into 5,6-dihydroxyindole-2-carboxylic acid (DHICA). A distinct enzyme activity isolated from bovine eye lens catalyzes the tautomerization of D-dopachrome, the non-physiological stereoisomer of the natural compound, L-dopachrome, to DHICA. (See FIG. 1). Purification and N-terminal sequence analysis of the protein responsible for this tautomerase activity for the non-naturally occurring substrate, D-dopachrome, identified the enzyme to be the bovine homolog of MIF. The enzymatic activity of purified, native MIF was confirmed by studies of recombinant human MIF, which was also found to catalyze selectively the same tautomerization reaction with D-dopachrome as substrate, but to be inactive toward L-dopachrome.
The present invention encompasses assays to identify compounds that inhibit the enzymatic activity of MIF which catalyzes the tautomerization of MIF-substrates, such as D-dopachrome to DHICA. In general, the assay is conducted in vitro by adding, mixing or combining MIF and a suitable substrate in the presence or absence of a test compound, and measuring the tautomerization of the substrate. The test compounds that inhibit tautomerization in the assay are identified as MIF inhibitors.
The present invention relates to the use of such identified inhibitors in pharmaceutical compositions designed to inhibit MIF biological activity for the treatment of cytokine toxicity-related disorders. The present invention encompasses the preparation of such pharmaceutical compositions and methods to use such compositions for the treatment of various conditions involving cytokine-mediated toxicity, which include, but are not limited to shock, inflammation, graft-versus-host disease, and/or autoimmune diseases.
3.1. Definitions
The following terms as used herein have the definitions indicated.
MIF refers to vertebrate forms of macrophage migration inhibitory factor and functional equivalents thereof, including but not limited to MIF protein and lysates containing MIF activity whether obtained from vertebrate tissue sources, including, but not limited to human, bovine, rat, mouse and chicken tissues, or from recombinant sources of MIF protein expressed by genetic engineering.
MIF tautomerase activity, MIF dopachrome tautomerase activity and MIF tautomerization activity all refer to the same enzymatic activity of MIF that is the ability of MIF to catalyze the tautomerization of a MIF-substrate.
MIF substrate refers to (a) D-dopachrome [D-3,5-dihydro-6-hydroxy-5-oxo-2H-indole-2-carboxylic acid], (b) D-dopachrome-methyl ester [D-3,5-dihydro-6-hydroxy-5-oxo-2H-indole-2-carboxylic acid methyl ester], or (c) L-dopachrome-methyl ester [L-3,5-dihydro-6-hydroxy-5-oxo-2H-indole-2-carboxylic acid methyl ester], or derivatives thereof.
Test compounds or test substances or test inhibitors refers to drugs, ligands (natural or synthetic), proteins, peptides or small organic molecules to be tested for their ability to inhibit the tautomerase activity of MIF.